Various "promoter" sequences are available which may be used in the genetic engineering of plants. Depending upon the transcription initiation characteristics desired (strength, tissue specificity, developmental specificity, etc.), different promoters are available which may be employed to initiate the transcription, and in some cases the translation, of a DNA sequence of interest joined at the 3' end of the promoter region.
For example, promoters, or "transcription and translation initiation regions," known as 35S Cauliflower Mosaic Virus (CaMV 35S), mannopine synthase (mas) and octopine synthase (ocs) have been used successfully to direct the expression of desired nucleic acid sequences in transformed plant tissue. The relative activities of these promoters may be ranked: CaMV 35S &gt;mas &gt;ocs. When expressed in a transgenic plant, DNA sequences under the control of these promoters are found at relatively low levels and expressed fairly evenly (i.e., constitutively) throughout the plant. A variation of the CaMV 35S promoter, known as the "double 35S promoter (D35S)" because of repeated CaMV 35S sequence engineered into the promoter, shows markedly stronger transcription initiation properties than the "unenhanced" CaMV 35S.
Transcription and translation initiation regions also have been developed from indigenous plant genes, especially when differentially specific characteristics are desired. One example of such a promoter is the "2AII promoter" described in WO 88/09334; a nucleic acid sequence under the regulatory control of the 5' non-coding region of the tomato 2AII gene will be preferentially transcribed in developing fruit tissue.
Of particular interest is the discovery of promoters which demonstrate enhanced transcription initiation characteristics in rapidly dividing cells or rapidly growing tissue, against stress or other detrimental factors. For example, the site of action of various herbicides is rapidly dividing cells. Insects frequently target young tender tissue for attack resulting in injury at this site. Certain plant diseases are particularly severe with young rapidly dividing cells. Also, the tender new tissue is most sensitive to stresses such as frost, so that enhanced production of products which protect against frost or inhibition of endogenous products which enhance the sensitivity to frost is of primary interest in such tissue.
Likewise, there are advantages with the increased expression of a DNA sequence of interest under the regulation of an inducible promoter. Such promoters may regulate the expression of genes in response to a variety of different environmental factors, such as light, wounding, exposure to heavy metals, and/or temperature, for example.
Heat stress in particular can be a problem in many useful agronomic crops. In the laboratory, heat shock is a useful tool to study the effect of a given DNA sequence under controlled conditions. Regulating a gene's activity by heat shock might also be used to control plant features for a given period of time to alter and/or control a biochemical pathway, express controlled amounts of otherwise toxic substances, or the like, in the heat shock affected tissues. Thus, the enhanced production of desired DNA sequences under the control of a heat shock induced promoter could allow for the genetic engineering of plants with improved heat tolerance for field use. Alternatively, heat induced promoter activity might be a useful "switch" for the induction of a desired characteristic for a given period of time under otherwise controlled conditions.
Plant stress may also be induced by wounding. As noted above, insects often selectively chew on young tissue. Wound stress can also occur in older, more mature tissue, as a result of breaking, cutting, chewing, boring, and the like. Some promoters are believed to display wound-inducible characteristics, although they are often selective to "crush" or "chew" type wounds and do not offer a wound response to "cutting" or "excision" type wounds. Broad spectrum wound promoters are desired.
Thus, the discovery of new promoters with useful transcript initiation patterns, especially ones having very strong promoter activity, are desired for the controlled expression of desired nucleic acid sequences. Promoters which show enhanced activity in rapidly dividing tissue and/or show enhanced activity induced by environmental phenomena are of special interest for many genetic engineering applications in plant tissue. Protein sequences associated with such promoters may also provide useful genetic engineering tools to enhance plant characteristics generally, as these critical gene sequences are abundant in young or stressed plant tissue.
In addition, sequence enhancers which could confer high expression to plant or animal DNA sequences associated therewith are desired to improve expression levels of flanking sequences, generally. In some non-plant species, particular sequences found flanking certain genes have been postulated to bind to the "nuclear scaffold."
The nuclear scaffold is comprised of non-histone proteins, such as topoisomerase II (Earnshaw, et al., J. Cell Biol. (1985) 100:1706-1715). Chromatin loops are attached to the scaffold at specific DNA sequences (Gasser & Laemmli, Cell (1986) 46:521-530) located at the base of the loops. Attachment sites are found in both 5' and 3' untranslated regions.